This exploratory R21 proposal focuses on evaluating two Reticulocyte Binding-Like (RBL) protein vaccine candidates in rhesus monkeys with a blood-stage challenge to test for efficacy, providing for the first-time a proof of principle for this category of merozoite vaccine antigens, and also preparing P. vivax RBL antigens for future pre-clinical trials. Plasmodium knowlesi blood-stage infections in rhesus macaques become lethal 1-2 weeks after inoculation unless treated with anti-malarial drugs. We are proposing to test the potential of P. knowlesi RBL binding domains to function as immunogens that will attenuate the usual lethality of this parasite. If protection can be achieved against a conserved key biological target in monkeys, like the RBLs, there can be heightened confidence for advancing the homologous P. falciparum and P. vivax target(s) as a vaccine(s) in clinical trials. This project builds upon this group's discovery of the P. vivax Reticulocyte Binding Proteins (PvRBPs), the subsequent identification of these proteins in other species, (P. falciparum, P. cynomolgi, P. coatneyi, P. reichenowi, and P. knowlesi), and the identification and preparation as immunogens of two P. knowlesi RBL binding domains. The RBL proteins (also known as the reticulocyte-binding homologue (RH) protein superfamily) are located at the apical pole of merozoites, bind erythrocytes and are believed to be instrumental in directing merozoite invasion. Multiple rbl genes exist in each Plasmodium species (with only 2 in P. knowlesi, yet as many as 14 in P. yoelii), and data suggests that each species has maintained the ability to use one or another of the encoded ligands, or possibly combinations, as adhesion molecules to ensure entry into red blood cells. Critically, each species appears to have at least two RBLs that are essential for merozoite invasion. In Aim 1 using a rhesus monkey model we propose to test the hypothesis that immunization with an RBL red blood cell binding domain can limit a lethal infection, and that the combination of multiple RBL binding domain regions will provide greater protection. In Aim 2, we will test the hypothesis that antisera to homologous binding domain regions from P. vivax RBL proteins will inhibit invasion of merozoites in vitro, as a prelude to possible pre-clinical vaccine trials using these proteins in NHPs. We will work to optimize the expression and purification of homologous binding domain regions from expressed P. vivax RBL proteins, characterize functionality (i.e. binding), develop and test the ability of these RBLs or specific antisera to inhibit invasion in vitro. Regardless of the outcome from vaccine testing, we will have gained knowledge and additional tools to study the RBLs as critical ligands for red blood cell invasion and as possible biological targets of intervention. PUBLIC HEALTH RELEVANCE: A malaria vaccine would be extremely valuable tool to help control malaria. Novel approaches and new paradigms are needed that can expedite the testing and provide some form of validation for theoretically sound malaria vaccine candidates. This proposal is straightforward, while also being innovative, risky and exploratory. We will learn whether RBL-based malaria vaccines can provide protection against erythrocytic challenge and also develop valuable tools for advancing investigations relating to merozoite invasion of red blood cells. This research will also gain valuable information on the immune response to the RBLs and pave the way for potential pre- clinical malaria vaccine trials based on P. vivax RBL immunogens.